title: Effect of F8 B domain gene variants on synthesis, secretion, activity and stability of factor VIII protein.
abstract: The B domain of the coagulation factor (F)VIII comprises some unique characteristics. Though the B domain is important for processing, intracellular transport and secretion of FVIII protein, its role in the coagulation still remains unclear. This study aims to investigate the influence of 19 reported B domain variants on quantity and quality of expressed FVIII protein. F8 variants were transiently expressed in HEK293T cells. Media and cell lysates were collected after 72 hours. FVIII synthesis, relative secretion, activity and thermostability were analysed in comparison to FVIII wild-type. Eleven of 19 analysed B domain variants showed normal FVIII activity (FVIII:C), and antigen values (40-150 %). Eight variants exhibited a decreased FVIII:C, corresponding to a mild phenotype most likely due to impaired expression and secretion mechanism, reduced thermostability or combined mechanisms. One variant, p.His1066Tyr, showed markedly reduced FVIII antigen in cell lysate. The variants p.Asp845Glu, p.His998Gln, and p.Ala1610Ser revealed a significantly decreased relative secretion. Additionally, six B domain variants significantly reduced stability of FVIII. In conclusion, none of the analysed missense mutations was causative for a severe haemophilia A (HA) phenotype. Nevertheless, the mutations p.Asp845Glu, p.Pro947Arg, p.Glu1057Lys, p.His1066Tyr, p.Arg1126Trp, p.Arg1329His, p.Leu1481Pro, and p.Ala1610Ser resulted in decreased FVIII:C values that may explain mild HA phenotypes.
authors.forename: Saskia
authors.initials: S
authors.lastname: Pahl
authors.forename: Anna
authors.initials: A
authors.lastname: Pavlova
authors.forename: Julia
authors.initials: J
authors.lastname: Driesen
authors.affiliation: Prof. Dr. Johannes Oldenburg, Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Sigmund-Freud-Str. 25, Germany, Tel.: +49 228 287 15175, Fax: +49 228 287 14783, E-mail: johannes.oldenburg@ukb.uni-bonn.de.
authors.forename: Johannes
authors.initials: J
authors.lastname: Oldenburg
mesh_terms: D017398:Alternative Splicing; D001665:Binding Sites; D001777:Blood Coagulation / Q000235:genetics; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D056726:Genetic Association Studies; D014644:Genetic Variation; D057809:HEK293 Cells; D006467:Hemophilia A / Q000235:genetics / Q000503:physiopathology; D006358:Hot Temperature; D006801:Humans; D020125:Mutation, Missense; D010641:Phenotype; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D013696:Temperature
chemical_list: C078147:F8 protein, human; D005169:Factor VIII
keywords: B domain; Factor VIII; genotype-phenotype relationship; missense mutation